1. Field of the Invention
The invention relates to new effectors of dipeptidyl peptidase IV (DP IV) and methods of treatment comprising the topical application thereof. These effectors can be used for targeted influencing of locally limited pathophysiological and physiological processes (inflammation, chemotaxis, autoimmune diseases wound healing), wherein the enzymatic activity and binding activities of dipeptidyl peptidase IV and of enzymes having comparable or identical activity and of proteins having a related primary structure (e.g. FAP, Fibroblast Activation Protein (Levy et al., 1999)) are influenced by means of effectors (substrates, pseudo-substrates, inhibitors, antibodies, binding proteins, binding antagonists, binding agonists, inter alia).
2. Related Art
In addition to proteases involved in non-specific proteolysis, which results, finally, in the breakdown of proteins into amino acids, regulatory proteases are known, which take part in the functionalization (activation, deactivation, modification) of endogenous peptide active substances (Kirschke et al., 1995; Kräusslich and Wimmer, 1987). Especially in the context of immunological research and neuropeptide research, a number of such so-called convertases, signal peptidases or enkephalinases have been discovered (Gomez et al., 1988; Ansorge et al., 1991). Because of the frequency of the presence of the amino acid proline in a multiplicity of peptide hormones and because of the related structural properties of those peptides, a function analogous to the signal peptidases is being discussed for proline-specific peptidases (Yaron and Naider, 1993; Vanhoof et al. 1995). As a result of its particular structure, proline in those peptides determines both the conformation and stability of those peptides, protecting them from breakdown by non-specific proteases (Kessler, 1982). Enzymes that, in contrast, act in highly specific, structure-modifying manner on proline-containing sequences (HIV-protease, cyclophilin, inter alia) are attractive targets for current active substance research. In particular, for the peptidases prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DP IV), which cleave after the proline, it has been possible to conclude that there probably are connections between modification of the biological activity of natural peptide substrates and selective cleavage thereof by those enzymes. It is accordingly postulated that PEP plays a part in learning and in the memory process and that DP IV is involved in signal transmission during the immune response (Ishiura et al., 1989; Hegen et al., 1990).
DP IV activity and DP IV-analogous activity (for example, the lysosomal DP II has a substrate specificity that is almost identical to DP IV) is to be found in the bloodstream and in almost all organs, where it cleaves dipeptides from the N terminus of biologically active peptides with high specificity when their sequence contains proline or alanine as residues adjacent to the N-terminal amino acid. It is therefore assumed that this enzyme is involved in regulating the biological activity of polypeptides in vivo (Vanhoof et al., 1995).
It has recently been shown that a series of chemokines (RANTES, SDF-1 alpha, MDC, eotaxin, inter alia) are substrates of DP IV and that they are modulated in their function by DP IV (Proost et al., 1998; Proost et al., 1998; Proost et al., 1999; Shioda et al., 1998). As a result of their chemotactic action, chemokines are substantially involved in the regulation of local immunological processes, such as autoimmune processes, inflammation and wound healing (Nelson and Krensky, 1998). In more recent work, we have been able to demonstrate that biologically active peptides having serine or threonine in the P1-position (glucagon, VIP, PACAP) are also substrates of DP IV.
A series of biologically active DP IV-substrates (substance P, somatostatin, VIP, PACAP, inter alia) are involved in the regulation of neuronal, immunological and vasoactive processes in the skin (Scholzen et al., 1998); (Wallengren, 1997). Dipeptidyl peptidase IV accordingly represents an important control centre in regulating the activity of gastrointestinally, immunologically and neurologically active peptides and, consequently, is an interesting therapeutic target (Augustyns et al., 1999). The precise details of the signal cascades have not, however, been clarified fully.
The role of DP IV in the regulation of blood sugar is known in greater detail. As a result of limited proteolysis, the incretins GIP1-4 and GLP-17-37 are inactivated. Inhibition of plasma-DP IV activity leads, by way of prolonged activity of the incretins and increased insulin release, to normalization of the blood sugar level (Demuth et al., 1996; Pauly et al., 1996; Pauly et al., 1999).
The role of DP IV in the immune system has not yet been fully clarified. It is an activation marker of T-lymphocytes and a receptor for adenosinedeaminase. The use of DP IV-inhibitors has immunosuppressant effects in cell culture and in vivo (Ansorge et al., 1995; Reinhold et al., 1997; Kubota et al., 1992). Using monoclonal antibodies against CD26, stimulatory effects on intracellular signal cascades (Ca2+ influx, kinase activations) have been obtained, in some cases independently of the enzymatic activity of the enzyme (Hegen et al., 1993; Kameoka et al, 1995; Tanaka et al., 1993; Kähne et al., 1995).
Lysyl-prolyl analogues derived from the N-terminal sequence of substance P have shown a wound-healing-promoting effect, which is attributed to the structural similarity to substance P. In contrast, irreversible DP IV-inhibitors used systemically have resulted in inhibition of wound healing (Buntroek et al., 1988; Kohl et al., 1991; Kohl et al., 1989).
In addition to the use of DP IV-inhibitors for the normalization of blood glucose, DP IV-inhibitors have hitherto been used systemically for treating arthritis in an animal model.
In arthritis patients and in animal arthritis models, a reduction in DP IV activity has been observed (Küllertz and Boigk, 1986; Fujita et al., 1992). In particular, as a result of oral or subcutaneous administration of systemically acting DP IV-inhibitors, suppression of alkyldiamine-induced arthritis has been achieved in an animal model (Tanaka et al., 1997; Tanaka et al., 1998).
In relation to other autoimmune diseases as well, an effect has been obtained using DP IV-inhibitors. For example, as a result of DP IV inhibition it has been possible to achieve suppression of the proliferation of myelin basic protein-specific T cell clones (Reinhold et al., 1998).
In the case of various skin diseases (psoriasis, lichen planus) and cancerigenic diseases of the skin, it has been possible to demonstrate increased DP IV activity in keratinocytes and fibroblasts (Novelli et al., 1996; Raynaud et al., 1992).
Fibroblast activation protein, which is closely related to DP IV, having approximately 50% sequence homology with respect to DP IV, and which is probably the same as the seprase described by Piñieiro-Sanchez et al., 1997, is also expressed to an increased extent by inactivated fibroblasts of epithelial carcinomas and healing wounds (Niedermeyer et al., 1998).
Because of the wide distribution of the protein in the body and the wide variety of mechanisms involving DP IV, DP IV activities and DP IV-related proteins, systemic therapy (enteral or parenteral administration) with DP IV-inhibitors can result in a series of undesirable side-effects. For example, parenteral or enteral administration of DP IV-inhibitors will intervene in a regulating or deregulating manner in glucose metabolism.
It has now been possible to show that side chain-modified substrates of the enzyme dipeptidyl peptidase IV can be recognized by the enzyme and cleaved in the same way as unmodified substrates (Demuth, H.-U., Heins, J., 1995).
For example, it has been possible to show that phosphorylated dipeptide-(B)-p-nitroanilides (KASPARI, A, et al., 1996) are substrates of DP IV. DP IV-inhibitors such as, for example, Glu(Gly)-Thia or Lys(Z-NO2)-Thia (Reinhold, D., et al., 1998) are transported completely.